ES2553189T3 - Preparation of nucleotide oligomer - Google Patents

Abstract

A method of preparing ribonucleotide oligomers, comprising: (a) coupling a ribonucleotide dimer or ribonucleotide trimer to a ribonucleoside bound to solid supports or to universal solid supports as a starting material; (b) sequentially coupling ribonucleotide monomers to the structures resulting from step (a) to prepare ribonucleotide oligomers; and (c) removing ribonucleotide oligomers from solid supports. wherein the ribonucleotide dimer in step (a) is represented by Formula 5: ** Formula ** wherein each R1, R2, R3 and R5 are independently protecting groups, each B1 and B2 are independently ** Formula * * nucleoside bases, and R6 is hydrogen or where iPr is isopropyl; wherein the ribonucleotide trimer in step (a) is represented by Formula 6: ** Formula ** wherein each R1, R2, R3, R4, R5, and R6 are independently protecting groups, each B1, B2, and B3 are ** Formula ** independently nucleoside bases, and R7 is hydrogen or where iPr is isopropyl.

Description

DESCRIPTION

Preparation of nucleotide oligomer 5 Technical field

The present invention relates to a method for preparing nucleotide oligomers. More specifically, the present invention relates to solid phase synthesis of oligoribonucleotides.

10 Background of the technique

A variety of techniques for the preparation of nucleotide oligomers are known.

For example, methods of preparing nucleotide oligomers can be found in the following references: Khorana et al., J. Molec. Biol. 72: 209 (1972); Reese, Tetrahedron Lett. 34: 3143 (1978); Beaucage and Caruthers, Tetrahedron Lett. 22: 1859 (1981); US Patent No. 5,149,798; Agrawal and Goodchild, Tetrahedron Lett. 28: 3539 (1987); Connolly et al. Biochemistry 23, 3443 (1984); Jager et al., Biochemistry 27: 7237 (1988): Agrawal et al. Proc. Nati Acad. Sci. USA 85: 7079 (1988), for example, Methods in Molecular Biology, vol. 20, Protocols for Oligonucleotides and Analogs, pp. 63-80 (S. Agrawal, Ed, Humana Press 1993); Methods in Molecular Biology, vol. 20 26: Protocols for Oligonucleotide Conjugates (Agrawal Ed., Humana Press, Totowa, N.J. 1994); Oligonucleotides and Analogues: A Practical Approach pp. 155-183 (Eckstein, Ed., IRL Press, Oxford 1991); Antisense Res. And Applns. p 375 (Crooke and Lebleu, Eds., CRC Press, Boca Raton, Fia. 1993); and Gene Regulation: Biology of Antisense RNA and DNA (Erickson and Izant, eds., Raven Press, New York, 1992).

25 Antisense RNA hybridizes to nucleic acid molecules to result in inhibition of gene expression. Many researchers have reported the inhibition of specific gene expression or therapeutic feasibility of particular diseases through the use of antisense RNA (Barker et al. Proc. Nati. Acad. Sci. USA 93: 514 (1996); Agrawal et al. , Proc. Nati. Acad. Sci. USA 85: 7079 (1988); Letter et al., Proc. Nati. Acad. Sci. USA 87: 3420-3434 (1990); and Offensperger et al. EMBO J. 12: 1257 (1993)).

30

Meanwhile, RNA-mediated interference (RNA) is a phenomenon in which a small 21-25 nucleotide RNA fragment selectively binds to and degrades mRNA that has a complementary sequence, which suppresses protein expression suppression. (Shen C, et al., FEBS Lett. 539 (1-3): 111-4 (2003)). The phenomenon of RNA was first discovered in 1995 as part of the mechanism of gene regulation in Caenorphabditis 35 elegans and plants. In 1998, Dr. Andrew Fire of the Carnegie Institute of Washington and Dr. Craig Mello of the University of Massachusetts Medical School, and his team discovered experimentally that the expression of a specific gene can be significantly inhibited when double stranded RNA (cRNA) ) corresponding to a base sequence of the specific gene is injected in C. elegans (Fire A. et al., Nature. 391 (6669): 806-11 (1998)). The long chain mRNA injected into C. elegans is cleaved into a short double stranded RNA fragment called small interference RNA (pRNA) approximately 21-25 bp long, by the enzymatic action of Dicer belonging to a member of the RNase III family of nucleases that specifically cleave double stranded RNA. The resulting short rRNA is then incorporated into the RNA-induced silencing complex (RISC), in which the siRNA hybrid is unwound into two strands. Subsequently, the single stranded mRNA binds to a specific gene mRNA with a complementary sequence and makes it intra-perceived, thereby inhibiting the expression of the corresponding gene. In addition, Elbashir and his companions have reported that the expression of a specific gene can be selectively inhibited by injection of short rRNA (rRNA) consisting of 21 bases of cultured mammalian cells, this finding leads to significant increases in the practical applicability of RNA in mammalian cells (Elbashir, SM et al. Nature 411 (6836): 494-8 (2001)).

fifty

At this time, techniques of inhibition of gene expression mediated by siRNA are widely used in the functional understanding of various genes and a large amount of research has been actively focused on the exploitation of these siRNAs for the development of therapeutic agents for the treatment of intractable diseases such as cancers, infectious diseases, etc. (Moldy Sioud. Therapeutic siRNAs, Trends in 55 pharmacological Sciences 2004; 22-28).

As noted previously, many attempts have been made to develop therapeutic agents or diagnostic agents using antisense RNA and siRNA. To this end, there is an urgent need for an efficient mass production project of oligoribonucleotides.

60

The synthesis of nucleotide oligomers is usually carried out by sequential coupling of monomer units in solid resins using an automatic DNA / RNA synthesizer (or oligonucleotide). DNA oligomers can be synthesized with good performance. On the contrary, the synthesis of RNA oligomers, for example, ribonucleotide oligomers involve several disadvantages due to the steric hindrance of a protective group of a 2'-OH group, such as long synthesis period and low coupling efficiency that entails a

Low production performance making it difficult to obtain high purity RNA oligos.

Disclosure of the invention 5 Technical problem

As a result of a variety of extensive and intensive studies and experiments to solve the problems that have been previously described and to find a method that is capable of achieving high purity and practical production of oligomeric species such as nucleotide oligomers, in particular ribonucleotide oligomers or 10 small interfering RNA (siRNA), the inventors of the present invention found that it is possible to achieve the production of ribonucleotide oligomers with significantly improved purity by using a ribonucleotide dimer or ribonucleotide trimer as the first nucleotide synton that will bind to solid supports. The present invention has been completed based on these findings.

Therefore, the object of the present invention is to provide an oligomer preparation method.

ribonucleotides

Technical solution

The impurities produced during the synthesis of nucleotide oligomers are mainly composed of short sequences that have a lower degree of coupling than full length sequences (Nmers), and are usually expressed as (N-1) mers, (N- 2) mers, (Nx) mers, or the like. Impurity of oligomers shorter than full-length Nmers generally occurs due to incomplete addition of the cap at a stage of adding the product cap followed by the coupling reaction, after coupling 25 of nucleotide units to supports solid.

In addition, the species of impurities that are more difficult to separate during purification of desired nucleotide oligomers are (N-I) mere eluting in a position close to that of the desired oligomers in chromatograms.

30

One study showed that the content of (N-1) -mer and phosphodiester could be reduced by the use of dimeric phosphoramidite syntheses compared to the use of monomeric phosphoramidites (Krotz AH et al., Bioorganic Medicinal Chemistry Letters, 1997, vol 7 , No. 1, pages 73-78).

35 A subsequent study showed that the content of (N-1) -mer could be reduced by the use of trimeric phosphoramidite syntheses compared to the use of monomeric phosphoramidites (Eleuteri A, et al., Nucleosides & Nucleotides, 1999, 18 ( 3), pages 475-483).

However, according to the present invention that uses a dimer or trimer, not a monomer, in the first coupling reaction 40, cases of (N-1) mers that are difficult to eliminate during the purification process and of this are avoided. In this way, pure ribonucleotide oligomers are easily obtained with the formation of (N-2) mers or (N-3) mers easily purifiable. In particular, when it is desired to use nucleotide oligomers as therapeutic, they are purified by chromatography techniques. In this regard, the Nmers and (N-1) mers elute over a very close time interval, so it is difficult to successfully achieve the chromatographic separation of (N-45 1) mers. However, when a ribonucleotide dimer or trimer is used as the first nucleotide block that is coupled to solid supports, as disclosed in the present invention, the formation of impurities of (N-1) mer is significantly reduced, thereby generating improvements. pronounced purification yields and consequently a significant reduction in production costs.

As will be demonstrated in the Examples hereafter, the present invention allows the reduction of (N-1) mers that occurs largely in the first coupling reaction, as well as the general decrease in impurities of (Nx) mer . It is believed that this is because when the coupling of a longer dimer or trimer is performed instead of a monomer in the first coupling reaction on solid supports, the next binding of a monomer to the coupled dimer or trimer is much more spatially advantageous that the binding of a next monomer to a capless site, which consequently decreases the formation of oligomers that have a shorter sequence length than the desired oligomer.

In addition, the present invention provides the following differences and excellent effects compared to the conventional technique (WO 02/20543).

60

1) The conventional technique employs only dimers for the synthesis of nucleotide oligomers and therefore prepares the nucleotide oligomers of a repetitive sequence in the dimer, while the present invention relates to the preparation of rinucleotide oligomers, which involves the use of a dimer or trimer unit only in the first coupling reaction on solid supports. That is, the conventional technique requires various types of dimers of up to 10 types when it is desired to prepare siRNA oligomers using dimer units. In

In other words, it is necessary to synthesize 10 types of dimers for this purpose, so long-term periods of synthesis and high production costs are required. On the contrary, the present invention employs only a kind of dimer or trimer in the first coupling stage and then low-cost monomer units common in the later stages, which allows the production of high purity and low cost of rinucleotide oligomers.

5

2) In addition, the present invention achieves a shorter synthesis time than the conventional technique. Typically, a coupling reaction of the RNA nucleotide oligomer synthesis takes 10 times longer than the synthesis of the DNA nucleotide oligomer. In this regard, although the conventional technique deals with an example of DNA nucleotide oligomer synthesis, it was suggested that a coupling reaction of 10 dimers in the DNA requires a period of 20 to 60 min. On the other hand, according to the present invention, the coupling of the first dimer for the synthesis of RNA nucleotide oligomers requires a period of 10 to 20 min and the coupling of the subsequent monomer takes 10 min, so the total synthesis time is long. shorter than conventional technique. As a consequence, the present invention shortens the production periods of the products to significantly reduce production costs when nucleotide oligomers are formulated in therapeutics.

The present invention provides a method of preparing ribonucleotide oligomers, comprising:

(a) coupling a ribonucleotide dimer or ribonucleotide trimer to a ribonucleoside attached to solid supports or to universal solid supports as a starting material;

(b) sequentially coupling ribonucleotide monomers to the structures resulting from step (a) to prepare ribonucleotide oligomers; Y

(c) remove ribonucleotide oligomers from solid supports.

In which the ribonucleotide dimer in step (a) is represented by Formula 5:

image 1

R3O — p = o

image2

(5)

in which each R1, R2, R3 and R5 are independently protecting groups, each B1 and B2 are independently

0 (CH2) 2CN

- <. ,

N (iPr) 2

nucleobases, and R6 is hydrogen or in which iPr is isopropyl;

In which ribonucleotide trimer in step (a) is represented by Formula 6:

image3

or or2

R ^ O '

-P = 0

I

or-

or,

B '

r5o-

0 OR4

one

P = 0

or

\

or.

Bi

OR7OR6

(6)

5

in which each R-i, R2, R3, R4, R5 and R6 are independently protecting groups, each B-i, B2 and B3 are

independently nucleobases, and R7 is hydrogen or isopropyl.

^ (CHafeCN

XN (iPr) 2

in which iPr is

In one embodiment of the present invention, the method of preparing ribonucleotide oligomers includes the steps of:

(a) coupling a ribonucleotide dimer according to Formula 5 prior to a ribonucleoside attached to solid supports or to 10 universal solid supports as starting material;

(b) sequentially coupling ribonucleotide monomers to the structures resulting from step (a) to prepare ribonucleotide oligomers; Y

(c) remove ribonucleotide oligomers from solid supports.

In another embodiment of the present invention, the method of preparing ribonucleotide oligomers includes the steps of:

(a) coupling a ribonucleotide trimer according to Formula 6 prior to a ribonucleoside attached to solid supports or 5 to universal solid supports as starting material;

(b) sequentially coupling ribonucleotide monomers to the structures resulting from step (a) to prepare ribonucleotide oligomers; Y

(c) remove ribonucleotide oligomers from solid supports.

10 As used herein, unless otherwise specified, the term "nucleotide" is intended to encompass ribonucleotides, deoxyribonucleotides and their derivatives.

As used herein, the term "ribonucleotide" refers to a nucleotide that does not have 2H-H of a carbon atom in position 2 of the sugar, and is intended to encompass naturally occurring ribonucleotides as well as their analogs. . In the context of the present invention, for example, the term "ribonucleotide" also encompasses ribonucleotide derivatives in which the alkyl (eg, methyl or ethyl) binds to -OH in the C2 carbon of the sugar or to a halogen atom (for example, fluorine) or binds to an amino group instead of -OH in the C2 sugar of sugar.

The term "deoxlribonucleotide" refers to a nucleotide containing 2H-H sugar, and is intended to encompass naturally occurring deoxyribonucleotides as well as their analogs.

Examples of nucleotide include modified nucleotides in the skeleton such as DNA or RNA phosphorothioate, DNA or RNA phosphorodithioate, and DNA or RNA phosphoramidate; sugar modified nucleotides such as 2'-0-methyl RNA, 2'-0-ethyl RNA, 2'-0-methoxyethyl RNA, 2'-fluoro RNA, 2'-halogen RNA, 2'-amino RNA, 2 '-0-alkyl RNA, 2'-0-alkoxy RNA, 2-O-alkyl DNA, 2'-0-alkynyl DNA, 2'-0-alkynyl DNA, hexose DNA, pyranosyl RNA, anhydrohexitol DNA, and nucleic acid blocked (ANB); and modified nucleotides in the base comprising a base such as C-5 substituted pyrimidines (substituents include fluoro-, bromo-, chloro-, iodine-, methyl-, ethyl-, vinyl-, formyl, ethynyl-, propynyl- , alkynyl, thiazolyl-, imidazolyl-, and pyridyl-), 7-deazapurines with C-7 substituents (substituents include fluoro-, bromo-, chloro-, iodine-, methyl-, ethyl-, vinyl-, formyl- , alkynyl-, alkenyl-, thiazolyl-, imidazolyl-, and pyridyl-), inosine and diaminopurine.

The nucleotide of the present invention is a ribonucleotide, preferably a phosphoramidite ribonucleoside.

As described herein, the nucleotide oligomer may include various types of nucleotide oligomers, for example, deoxyribonucleotide oligomers and their derivatives. As described and / or claimed herein, the nucleotide oligomer may be a naturally occurring nucleotide oligomer as well as a modified nucleotide oligomer. For example, one can cite modified nucleotide oligomers in the skeleton, such as DNA or RNA phosphorothioate, DNA or RNA phosphorodithioate and DNA or RNA phosphoramidate; Sugar-modified nucleotide oligomers such as 2-O-methyl RNA, 2-O-ethyl RNA, 2'-0-methoxyethyl RNA, 2C-fluoro RNA, 2'-halogen RNA, 2'-amino RNA, 2-O -alkyl RNA, 2-O-alkoxy RNA, 2-O-alkyl DNA, 2-O-allyl DNA, 2-O-alkynyl DNA, hexose DNA, pyranosyl RNA, anhydrohexitol DNA, and blocked nucleic acid (ANB); and base modified nucleotide oligomers, such as C-5 substituted pyrimidines (substituents include fluoro-, bromo-, chloro-, iodine-, methyl-, ethyl-, vinyl-, formyl-, ethynyl-, propynyl-, alkynyl-, thiazolyl-, imidazolyl-, and pyridyl-), 7-deazapurine with C-7 substituents (substituents include fluoro-, bromo-, chloro-, iodine-, methyl-, ethyl-, vinyl-, formyl- , alkynyl-, alkenyl-, thiazolyl-, imidazolyl-, and pyridyl-), inosine and diaminopurine.

The nucleotide oligomer of the present invention is a ribonucleotide oligomer.

Preferably, the ribonucleotide oligomer is one that contains at least one ribonucleotide selected from 2-O-halogen ribonucleotide, 2'-amino ribonucleotide, 2-O-alkyl ribonucleotide and 2-O-alkoxy ribonucleotide.

The present invention employs the ribonucleotide dimer or ribonucleotide trimer as the first coupling reagent that will bind to solid supports. Depending on the types of ribonucleotides located at the 3H-55 terminal end corresponding to the third sugar carbon, for example, the types of ribonucleotides attached to solid supports, the method of the present invention can be classified into 3 types of the following way:

1) The first is a case in which the ribonucleoside is located at the 3D-terminal end. That is, the ribonucleotide dimer or ribonucleotide trimer as the first coupling reagent is coupled to solid supports in

Those that a ribonucleoside monomer was preloaded as a starting material of a synthesis process, followed by sequential coupling of ribonucleotide monomers in the resulting structure to thereby prepare a ribonucleotide oligomer having a desired sequence.

2) The second is a case in which the ribonucleotide dimer is located at the 3H-terminal end. This case uses 65 universal solid supports as the starting material. Universal solid supports as starting material are

they are used in the first stage of the synthesis process, and the dibbonucleotide dimer is used as the first coupling reagent. Hereinafter, the carbononucleotide monomers are sequentially coupled to the resulting structure to thereby prepare a carbononucleotide oligomer having a desired sequence.

5 3) The third one is a case in which the rbonbonucleotide trimer is located at the 3ll-terminal end. This case also uses universal solid supports as starting material. Universal solid supports as starting material are used in the first stage of the synthesis process, and the ribonucleotide trimer is used as the first coupling reagent. Hereinafter, ribonucleotide monomers are sequentially coupled to the resulting structure to thereby prepare a ribonucleotide oligomer having a desired sequence.

10

The most preferred method of the three previously mentioned methods is a method in which the solid supports to which a ribonucleoside is previously attached is used as a starting material and the ribonucleotide dimer or trimer as the first coupling reagent is coupled to the ribonucleoside preloaded

As used herein, the term "universal solid supports" refers to solid supports that are free of a nucleoside or nucleotide oligomer covalently bonded thereto. Unlike pre-loaded supports, the use of universal solid supports allows the synthesis of any nucleotide oligomer regardless of the types of terminal sequences of nucleotide oligomers. When universal supports are used, a terminal sequence of the final synthetic nucleotide oligomer is determined by a

20 nucleotide tuner applied to the first coupling reaction of the synthesis of the nucleotide oligomer.

The present invention is practiced according to solid phase synthesis.

When the process of the present invention is carried out according to solid phase synthesis, an embodiment

Preferred of the present invention includes the following steps:

(a) coupling a nucleotide dimer [(NMP) 2] or nucleotide trimer [(NMP) 3] to [(NS) -i] of a nucleoside in the solid support [SS (NS) i] to prepare SS- (NS ) i- (NMP) 2 or SS- (NS) i- (NMP) 3;

(b) sequentially coupling nucleotide monomers to the structure resulting from step (a) to prepare

30 an SS- (NS) i- (NMP) 2- (NMP) n-3 or SS- (NS) i- (NMP) 3- (NMP) n-4; Y

(c) remove solid supports (SS) from the structure SS- (NS) i- (NMP) 2- (NMP) n-3 or SS- (NS) i- (NMP) 3- (NMP) n-4 to obtain a (NMP) „, in which the nucleotide is a ribonucleotide, the nucleoside is a ribonucleoside and the nucleotide dimer or trimer is a ribonucleotide dimer or trimer according to Formula 5 or 6, respectively.

When the nucleotide dimer [(NMP) 2] or nucleotide trimer [(NMP) 3] is coupled in the first stage to the solid supports to which a nucleoside monomer is previously attached, and the nucleotide monomers are then coupled in a manner sequential thereto, a nucleotide oligomer molecule with significantly improved purity can be prepared.

The nucleoside in the solid support [SS- (NS) i] is a structure in which a molecule of ribonucleoside or deoxyribonucleoside binds to solid supports. The solid supports can be any one of those used in the solid phase synthesis of nucleotide molecules. Alternatively, universal solid supports to which the ribonucleoside or deoxyribonucleoside does not bind can also be used. Preferably, such solid supports should have the following properties: (i) substantially without solubility

45 in the reagents used for the synthesis of the nucleotide oligomer, (ii) chemical stability against the reagents used for the synthesis of the nucleotide oligomer, (iii) feasibility of chemical modifications, (iv) loading capacity of desired nucleotide oligomers, (v) reasonable compressive strength to withstand the increase in pressure during the synthesis process, and (vi) desired particle size and distribution.

A material that can be used as solid supports in the present invention may preferably be an inorganic polymer and includes, for example, silica, porous glass, aluminum silicate, polystyrene, polyvinyl alcohol, polyvinyl acetate, borosilicate, metal oxide (such such as alumina and nickel oxide) and clay. More preferably, the solid supports for use in the present invention are controlled pore glass (VPC) and polystyrene.

The present invention employs a [SS- (NS) i] in which a nucleoside is previously attached to a surface of the solid supports, specifically a [SS- (rNS) i] in which a ribonucleoside binds to a surface of solid supports. The nucleoside is conventionally bound to solid supports through a 3'-OH group of sugar.

The coupling of the ribonucleotide dimer or ribonucleotide trimer to the ribonucleoside can be carried out by

60 various methods known in the art. For example, the details of the coupling method can be found in the following literature: US Patent No. 4,458,066 and US Patent No. 4,415,732; Caruthers et al., Genetic Engineeríng, 4: 1-17 (1982); and Users Manual Model 392 and 394 Polynucleotide Synthesizers, pages 6-1 through 6-22, Applied Biosystems, Part No. 901237 (1991).

Preferably, the coupling process is carried out according to a phosphoramidite method. For example, it

You can perform as follows. A phosphoramidite derivative of the ribonucleotide dimer or ribonucleotide trimer is added to the ribonucleoside while adding an activator, for example a weak acid (such as tetrazole, 5-ethylthiotetrazole, benzylthiotetrazole, etc.). More preferably, the usable activator is 5-ethylthiotetrazole. The addition of the weak acid leads to the formation of a reaction intermediate through the protonation of the phosphoramidite nitrogen. This is followed by the addition of the cap of the resulting product. The cap is added preferably with an acetic anhydride and 1-methylimidazole. Next, the capped product is oxidized using an oxidant such as iodine so that an internucleotide bond becomes a more stable phosphodiester of labile phosphite. The order of the stages of adding the hood and oxidation can be reversed. After the oxidation step, a hydroxyl protecting group is removed using a protic acid, for example, trichloroacetic acid or dichloroacetic acid.

The ribonucleotide dimer or ribonucleotide trimer of the present invention may have various types of bonds, preferably phosphodiester, phosphoramidate, alkyl phosphoramidate, alkyl phosphonate, phosphorothioate, alkyl phosphotriester, or alkyl phosphonothioate bonds, more preferably phosphodiester or phosphoramidate bonds.

fifteen

Preferably, the ribonucleotide dimer and the ribonucleotide trimer of the present invention are the ribonucleotide dimer of phosphoramidite and the ribonucleotide trimer of phosphoramidite, respectively.

Therefore, the ribonucleotide ollomer of the present invention has a phosphodiester, phosphoramidate, alkylphosphoramidate, alkyl phosphonate, phosphorothioate, alkylphosphotriester or alkyl phosphonothioate linkage, more preferably a phosphodiester or phosphoramidate bond.

According to the present invention, SS- (NS) i- (NMP) 2- (NMP) n-3 or SS- (NS) i- (NMP) 3- (NMP) n-4 with a desired sequence is finally prepared by sequential coupling of ribonucleotide monomers to the ribonucleotide dimer or ribonucleotide trimer bound to the nucleoside on the solid support [SS- (NS) i].

When ribonucleotide monomers are sequentially coupled, 5-ethylthiotetrazole is used as activator.

Finally, the desired product (NMP) n is obtained by removing the solid supports (SS) of SS- (NS) i- (NMP) 2- 30 (NMP) n-3 or SS- (NS) i- ( NMP) 3- (NMP) n-4. When universal solid supports without ribonucleoside binding are used, (NMP) n is obtained by removing solid supports (SS) from SS- (NMP) 2- (NMP) n-2 or SS- (NMP) 3- (NMP ) n-3

The removal of solid supports can be carried out by any conventional method known in the art. For example, solid supports can be removed using ammonium hydroxide.

35

According to the preferred embodiment of the present invention, the method of the present invention may further include a step of eliminating the protective groups attached to the ribonucleotide oligomer [(NMP) n], before or after step (c). The removal of the protecting groups can be carried out by any conventional method known in the art. For example, a phosphate protecting group can be removed by treating a solution of thiophenol or ammonium hydroxide, while benzoyl and isobutyryl groups attached to the base can be removed by heating the ribonucleotide oligomer in an ammonium hydroxide solution.

There is no particular limit on the length of the ribonucleotide oligomer [(NMP) n] prepared by the method of the present invention. Typically, the ribonucleotide oligomer is 10 to 50 nucleotides in length.

Four. Five

According to the method of the present invention, it is possible to efficiently synthesize a high purity oligoribonucleotide in a shorter period of time. The method of the present invention provides a ribonucleotide oligomer having a purity of 15-20% greater than the conventional technique.

In step (a) of the claimed method, it is preferred to employ the ribonucleotide dimer of Formula 5, wherein Ri is dimethoxytrityl; each R2 and Rs are t-butyl dimethylsilyl; and R3 is halogen substituted phenyl.

In addition, the method of the present invention comprises the additional step of preparing a ribonucleotide dimer comprising the coupling of a compound of Formula 1 and a compound of Formula 2:

image4

O- (1)

R40

or,

B1

OH ORs (2)

In Formulas 1 and 2, each R-i, R2, R3, R4 and R5 are independently protecting groups, and each B1 and B2 are independently nucleobases.

5 Examples of the protecting groups R1 and R4 in Formulas 1 and 2 may independently include, but are not limited to, dimethoxytrlthl, monomethoxytrlthl, trityl, and 9-phenyl-xanthen-9-yl (pixyl). Preferred examples of suitable groups for R2 and R5 may independently include, but are not limited to, t-butyl dimethylsilyl, tri-isopropyl silyloxymethyl (TOM), 1- (2-chloro ethoxyjet (CEE), 2-cyanoethoxymethyl (CEM ), bis (2-acetoxy) methyl (ACE), 1- (2- fluorophenyl) -4-methox¡p¡perid¡n-4-lo (Fpmp), 1- (4-chloro phenyl) -4- ethoxypiperidin-4-yl (CPEP), 1- [2-chloro-4-methyl) phenyl] -4- 10 methoxl-plperldln-4-llo (CTMP), 4-nltrophellethylsulfonyl (NPES), 4-chloro phenylethylsulfonyl (CPES) ), 1- (2-cyanoethoxy) ethyl (CNEE), trimethyl silyloxymethyl (SEM), methoxyethoxymethyl (MEM), levulinyl, 4-nitrophenylethyl (NPE), and 4-nitrophenylethyloxycarbonyl (NPEOC).

R3 is preferably halogen or carbobenzoxyl substituted phenyl, without wishing to be limited thereto. 15 Each of B1 and B2 is independently adenine, cytosine, guanine, uracil or a derivative thereof.

More preferably, in Formulas 1 and 2, R1 and R4 are dimethoxytrity, R2 and R5 are t-butyl dimethylsilyl, and R3 is halogen substituted phenyl (more preferably 2-chlorophenyl).

20 Each of B1 and B2 is a base to which a protective group joins or not. Examples of the base that can be located in B1 and B2 may include common bases, such as adenine, cytosine, guanine and uracil, as well as their derivatives. Preferably, base derivatives include xanthlene, hypoxanthine. 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halo, uracil and cytosine, 6-azo uracil and cytosine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, oxa, amino, thiol, thioalkyl and 25 hydroxyl adenines and guanines, 5-trifluoro-methyl uracils and cytosines, and 7-metllguanine or noslna.

The protecting group can join B1 and B2. Examples of the protecting group may include, but are not limited to, benzollo or isobutlryl, acetyl, dimethylformamidine (DMF), phenoxylacetyl (PAC) and its derivative, and 4-t-butylphenoxyacetyl (TAC).

30

As previously described, the reaction conditions for the coupling of the compound of Formula 1 to the compound of Formula 2 are the same as those of the coupling of the nucleotide dimer or trimer.

In addition, the method of the present invention comprises the additional step of preparing a carbonyl nucleotide dimer (rNMP) 2j, which comprises coupling a compound of Formula 3 and a compound of Formula 2:

image5

OH OR5

In Formulas 2 and 3, each R-i, R2, R3, R4 and Rs are independently protecting groups, and each B1 and B2 are independently nucleobases.

Preferably, examples of the protecting groups R1 and R4 may independently include, but are not limited to, dimethoxytrityl, monomethoxytrityl, trityl, and 9-phenyl-xanthen-9-yl (pixyl). Examples of suitable groups for R2 and R5 may independently include, but are not limited to, t-butyl dimethylsilyl, tri-isopropyl silyloxymethyl (TOM), 1- (2-chloro ethoxyjetyl (CEE), 2-cyanoethoxymethyl (CEM) , bis (2-acetoxy) methyl (ACE), 1- (2- fluorophenyl) -4-methoxypiperidin-4-yl (Fpmp), 1- (4-chloro-phenyl) -4-ethoxypiperidin-4-yl (CPEP ), 1- [2-Chloro-4-methyl) phenyl] -4-methoxy-piperidin-10 4-yl (Ctmp), 4-nitrophenylethylsulfonyl (NPES), 4-chloro phenylethylsulfonyl (CPES), 1- (2- cyanoethoxy) ethyl (CNEE), trimethyl sllllethoxymethyl (SEM), methoxletoxlmethyl (MEM), levulinyl, 4-nitrophenylethyl (NPE), and 4-nitrophenylethyloxycarbonyl (NPEOC). Examples of suitable groups for R31 may include, but are not limited to, cyanoalkyloxy (such as clanoethoxy and cyanomethoxy), 4-clano-2-butenylox, and dlfenylmethylsilethoxy. Non-limiting examples of suitable groups for R32 may include dlaqullamlno. Each B1 and B2 are independently adenine, cytosine, guanine and uracil, or their derivatives.

More preferably, in Formula 3, R1 is dimethoxytrityl, R2 is t-butyl dimethylsilyl, R31 is 2-cyanoethyloxy, and R32 is dlakylamino (more preferably diisopropylamino).

The coupling of the compound of Formula 2 to the compound of Formula 3 in the present invention can be carried out in the same manner indicated above, and a more preferred activator is 5-ethylthi-tetrazole.

In step (a) of the claimed method, it is preferred to employ the ribonucleotide trimer of Formula 6, wherein R1 is dimethoxytrityl; each R2, R4 and R6 are t-butyl dimethylsilyl; R3 is 2-cyanoethyl and R5 is halogen substituted phenyl.

In addition, the method of the present invention comprises the additional step of preparing a ribonucleotide trimer, comprising the steps of:

(a) reacting a ribonucleotide dimer of Formula 4 with an acid to remove R1 of Formula 4; Y

(b) coupling the product resulting from step (a) to a 3'-phosphoramidite ribonucleoside to prepare a ribonucleotide trimer.

image6

R 30

—P = 0

image7

OH OR5

(4)

In Formula 4, each R1, R2, R3, R4 and Rs are independently protecting groups, and each B1 and B2 are independently nucleobases.

5

In Formula 4, preferred examples of the protecting groups R1 may independently include, but are not limited to, dimethoxytrityl, monomethoxytrityl, trityl, and 9-phenyl-xanthen-9-yl (pixyl). Examples of suitable groups for R2 and Rs may independently include, but are not limited to, t-butyl dimethylsilyl, tri-isopropyl silyloxymethyl (TOM), 1- (2-chloro ethoxyjethyl (EEC), 2-cyanoethoxymethyl (CEM) , bis (2-acetoxy) methyl (ACE), 1- (2-fluorophenyl) -4- 10 methoxlplperldln-4-llo (Fpmp), 1- (4-chloro-phenyl) -4-ethoxypiperidin-4-yl ( Cpep), 1- [2-Chloro-4-methyl) phenyl] -4-methoxy-piperidin-4-yl (Ctmp), 4-nltrophellethylsulfonyl (NPES), 4-chloro phenylethylsulfonyl (CPES), 1- (2- cyanoethoxy) ethyl (CNEE), trimethyl silyloxymethyl (SEM), methoxyethoxymethyl (MEM), levulinyl, 4-nitrophenylethyl (NPE), and 4-nitrophenylethyloxycarbonyl (NPEOC). Examples of suitable groups for R3 may include, but are not limited to, hydrogen, and halogen substituted phenyl or carbobenzoxyl. Each B1 and B2 are independently adenine, cytosine, guanine, uracil or its derivatives.

More preferably, in Formula 4, R1 is dimethoxytrityl, R2 is t-butyl dimethylsilyl, R3 is hydrogen or halogen substituted phenyl (more preferably chlorophenyl), and R5 is tert-butyl dimethylsilyl.

The elimination of R1 from the carbonylotidic dimer of Formula 4 can be carried out by any conventional deprotection method known in the art, using a strong acid, for example, benzenesulfonic acid.

The coupling conditions of step (b) in the prior preparation method are the same as the conditions mentioned above.

25

Advantageous effects

The present invention allows efficient synthesis of high speed and high purity of ribonucleotide oligomers. The method of the present invention provides a ribonucleotide oligomer having a purity of 15-20% greater than the conventional technique.

Mode of the invention

The present invention will now be described in more detail with reference to the following Examples.

Hereinafter, the 31P NMR values measured are the values measured using Varian Mercury Plus 300 MHz.

[Reaction scheme 1]

RiO — i

OR,

Bi

0 OR2

one

RO — P = 0

OR'

3

image8

OH OR2

5 Synthesis of UpU, CpU and GpA ribonucleotide dimers (2a to 2c)

Ri = DMTr (dimethoxytrityl), R2 = TBDMS (tert-butyldimethylsilyl), R = o-chlorophenyl. 2a-Bi = U, B2 = U; 2b-Bi = bzC, B2 = U; 2c-Bi, = ibG, B2 = bzA; 3a-Bi = U; 3b-B-i, = bzC; 3c-Bi = ibG; 4a-B2 = U; 4b-B2 = bzA. bz = benzoyl, ib = isobutyryl.

Example 1: Synthesis of 5'-0- (dimethoxytrityl-2D-0-t-butyldimethylsilyluridine-3'-0- (2-chlorophenylphosphatol-5'-0-2'-0- (t-butyldimethylsilylhuridine (2a1)

Stage 1: Synthesis of 5'-Q- (dimethoxytrityl-2 -0-t-butyldimethylsilyluridine-3'-0- (2-chlorophenylphosphate1 (triethylammonium salt (3a1

fifteen

Triazole (0.63 g, 9.24 mmol, Sigma Aldrich) and anhydrous triethylamine (1.3 mL, 9.15 mmol, Sigma Aldrich) were dissolved in dioxane (20 mL), and the solution was cooled to 5 ° C . A solution of O-chlorophenyl phosphodichlorohydrate (1.1 g, 4.53 mmol, Sigma Aldrich) in 5 ml of dioxane was added dropwise to the resulting solution. After one hour, the mixed solution was filtered and added to S'-O-dimethoxyirityl ^ '- Ot-butyldimethylsilyluridine (2 g, 3.02 mmol) in 10 ml of pyridine which had cooled to -5 ° C . Then, 1-methylimidazole (0.38 ml, 4.6 mmol, Sigma Aldrich) was added thereto. After one hour, 0.1 M triethylammonium bicarbonate buffer (TEAB, 10 ml) was added to the previously cooled solution which was then concentrated. The residue was dissolved in dichloromethane (50 ml) and washed with 0.1 M TEAB (50 ml), and the aqueous layer was extracted twice with 20 ml of dichloromethane. The organic layer was collected, washed with 0.1 M TEAB (100 ml), and dried over sodium sulfate. The residue was concentrated using a vacuum pump to give 2.78 g (yield: 97%) of the title compound.

Stage 2: Synthesis of 5'-Q-fdimethoxytrityl-2 -0- (t-butyldimethylsilylhuridine-3'-0- (2-chlorophenylphosphate1-5'-Q-2'-0- (t-butyldimethylsilihuridine (2a1)

30 Triethylammonium salt of 5'-0- (d -methoxytritl-2T-0-t-but¡ld¡met¡ls¡luridine-3'-0- (2-chlorophenylphosphate) (3a, 1.47 g, 1.54 mmol) in Step 1 and 5'-0-dimethoxytrityl-2'-0-tert-butyldimethylsilyluridine (4a, 0.5 g, 1.4 mmol) was dissolved in 20 ml of pyridine and the solution was dried using a vacuum pump.1-Methylenesulfonyl-3-n -tro-1,2,4-triazole (MSNT, 0.68 g, 2.31 mmol, Sigma Aldrich) was added in 5 ml of fresh pyridine to the dried product The reaction solution was concentrated to approximately 3 ml, and 0.16 ml of 1-methylimidazole (1.89 mmol) was added thereto After 35 hours, the reaction solution it was cooled to 0 ° C and 2 ml of water were added thereto, the reaction solution was concentrated, the residual oil was dissolved in 15 ml of dichloromethane and washed with 15 ml of 0.1 M TEAB. The aqueous layer was washed with dichloromethane (3x5 ml.) The organic layer was collected and dried over sodium sulfate.The residue was purified by cr silica gel omatography to provide the title compound (0.68 g, yield: 41%). NMR 31P (DMSO), ppm: -6.38, -6.25 40

Example 2: Synthesis of 5'-0-dimethoxytrityl-N4-benzoyl-2l -0- (t-butyldimethylsilihcitidine-3'-0- (2-chlorophenylphosphate1-5'-0-2'-0- (t-butyldimethylsilihuridine ( 2b)

Stage 1: Synthesis of 5'-0-d -methoxytrityl-N4-benzoyl-2l -0- (t-butyldimethylsilylcitidine-3'-0- (2-chlorophenylphosphateUsal 45 of triethylammoniol Í3bí

Triazole (0.69 g, 10 mmol) and anhydrous triethylamine (1.4 mL, 9.9 mmol) were dissolved in dioxane (20 mL) and the solution was

cooled to 5 ° C. A solution of O-chlorophenyl phosphodichlorohydrate (1.2 g, 4.90 mmol) in 5 ml of dioxane was added dropwise to the resulting solution. After one hour, the mixed solution was filtered and added to 5'-O-dimethoxytrityl-N4-benzoyl-2'-0- (t-butyldimethylsilyl) citidine (2.5 g, 3.27 mmol, Sigma Aldrich) in 10 ml of anhydrous pyridine that had cooled to -5 ° C. Next, 1-methylimidazole (0.40 mL, 4.9 mmol, Sigma Aldrich) was added to the previous solution. 5 After one hour, 0.1 M TEAB (10 ml) was added to the cooled solution that was concentrated. The residue was dissolved in dichloromethane (50 ml) and washed with 0.1 M TEAB (50 ml). The aqueous layer was extracted twice with 20 ml of dichloromethane. The organic layer was collected, washed with 0.1 M TEAB (100 ml), dried over sodium sulfate and concentrated using a vacuum pump to give 3.08 g (yield: 94%) of the title compound.

10 Stage 2: Synthesis of 5'-0-dimethoxytrityl-N4-benzo¡l-2D-0- (t-butyldimethylsilyl1c¡d¡na-3'-0- (2-chlorophenphosphate1-5'- 0 -2'-0- (t-butyldimethylsilihuridine (2bl

5'-0-dimethoxytrityl-N4-benzoyl-2 l-0- (t-butyldimethylsilyl) citidine-3'-0- (2-chlorophenylphosphate) (triethylammonium salt) (3b, 3.24 g, 3, 07 mmol) in Step 1 and 5'-0-dimethoxytrityl-2ZI-0-tert-butyldimethylsilyluridine (4a, 1 g, 2.8) was dissolved

15 mmol) in 20 ml of pyridine, and the solution was dried using a vacuum pump. MSNT (1,364 g, 4.61 was added

mmol) in 10 ml of fresh pyridine to the dry product. The reaction solution was concentrated to approximately 3 ml and 0.25 ml of 1-methylimidazole (3.07 mmol) was added thereto. After one hour, the reaction solution was cooled to 0 ° C and 2 ml of water was added thereto. After the reaction solution was concentrated, the residual oil was dissolved in 15 ml of dichloromethane and washed with 15 ml of 0.1 M TEAB. The aqueous layer was washed with dichloromethane (3 × 5 ml), and the organic layer It was collected and dried over sodium sulfate. The residue was purified by silica gel chromatography to provide the title compound (1.47 g, yield: 40%).

NMR 31P (DMSO), 6ppm: -6.42, -6.10

Example 3: Synthesis of 5'-0-dimethoxytrityl-N2-isobutyryl-2D-0- (t-butyldimethylsilyl-yuanosine-3'-0-

Chlorophenylphosphate-5'-0-N4-benzoyl-2'-0- (t-butyldimethylsilihaden¡na <2c1

Stage 1: Synthesis of 5'-0-dimethoxytrityl-N2-isobutyryl-2D-0- (t-butyldimethylsilyltauanosine-3'-0- (2-

Chlorophenylphosphate Triethylammoniol salt (3cl

30 Triazole (1.37 g, 19.87 mmol) and anhydrous triethylamine (2.8 mL, 19.87 mmol) were dissolved in dioxane (20 mL) and the

solution was cooled to 5 ° C. A solution of O-chlorophenyl phosphodichlorohydrate (2.386 g, 9.74 was added dropwise

mmol) in 5 ml of dioxane to the resulting solution. After one hour, the mixed solution was filtered and added to S'-O-dimethoxytrritM-ISp-isobutirM ^ '- Ot-butyldimethylsMMguanosine (5 g. 6.5 mmol) in 10 ml of anhydrous pyridine which had cooled to -5 ° C. Then, 1-methylimidazole (0.80 ml, 9.74 mmol) was added thereto. After 35 hours, 0.1 M TEAB (10 ml) was added to the cooled solution which was then concentrated. The residue was dissolved in dichloromethane (50 ml) and washed with 0.1 M TEAB (50 ml) and the aqueous layer was extracted with dichloromethane (2 x 20 ml). The organic layer was collected, washed with 0.1 M TEAB (100 ml), dried over sodium sulfate and concentrated using a vacuum pump to give 6.55 g (yield: 95%) of the title compound.

40 Stage 2: Synthesis of 5'-0-d¡metox¡tr¡t¡l-N2-¡sobut¡r¡l-2D-0- (t-but¡ld¡met¡ls¡l¡hauanos¡na -3'-0- (2-

chlorophenphosphate1-5'-0-N4-benzo¡l-2'-0- (t-but¡ldimet¡ls¡l¡naden¡na (2c>

5l-0-dimethoxytrityl-N2-isobutyryl-2D-0- (t-butyldimethylsilyl) guanosine-3'-0- (2-chlorophenylphosphate) (triethylammonium salt) (3c, 1.34 g, 1.26 mmol was prepared ) in Step 1 and 5'-0-dimethoxytrityl-N4-benzoyl-2D-0-tert-butyldimethylsilyladenine (4b, 1 g, 2.8 mmol) was dissolved in 20 ml of pyridine, and the solution was dried using a vacuum pump MSNT (0.6 g, 1.89 mmol) in 10 ml of fresh pyridine was added to the dried product. The reaction solution was concentrated to approximately 3 ml and 0.16 ml of 1-methylimidazole (1.89 mmol) was added thereto. After 30 min, the reaction solution was cooled to 0 ° C and 2 ml of water was added thereto. The reaction solution was concentrated, and the residual oil was dissolved in 15 ml of dichloromethane and washed with 15 ml of 0.1 M TEAB. The aqueous layer was washed with dichloromethane (3 × 5 ml), and the organic layer was collected. and dried over sodium sulfate. The residue was purified by silica gel chromatography to provide the title compound (1,265 g, yield: 84%). NMR 31P (DMSO), ppm: -6.33, -6.14

[Reaction scheme 2]

image9

OH OR2

image10

5 Synthesis of ribonucleotide dimers UU, CU, GU and GA (2d to 2g).

Ri = DMTr (dimethoxytrityl), R2 = TBDMS, R = 2-cyanoethyl, 2d-Bi = U, B2 = U; 2e-Bi = U, B2 = bzC; 2f-Bi, = U, B2 = IbG; 2g-Bi = bzA; B2, = IbG; 4a-Bi = U; 4b-Bi = bzA. 5a-B2 = U, 5b-B2 = bzC, 5c-B2 = ibG.

10 Example 4: Synthesis of 5'-0-d¡metoxitrit¡l-2n-0- (t-but¡ld¡met¡ls¡l¡hur¡dil-3'-0-rcianoetoxifosfinol (3 '-> 5 'l-2'-Qt- butyldimetillsyliluridine (2d1

5, -dimethoxytrityl-uridine-2, -0-t-butyldimethylsyl-3, - [(2-cyanoethyl) - (N, N-diisopropyl)] phosphoramidite (5a, 1,085 g, 1) , 26 mmol) and S'-O-dimethoxytrityl ^ '- Ot-butyldimethylsilyluridine (4a, 0.3 g, 0.84 mmol) in 10 ml of anhydrous acetonitrile, and the solution was concentrated until it became gum. 5-Benzylltotetrazole (0.483 g, 2.52 mmol, ChemGene) was dissolved in 20 ml of acetonitrile, and the solution was concentrated until crystals formed. Two solutions were combined using 20 ml of acetonitrile and concentrated to 3 ml. After one hour, the combined solution was cooled to 0 ° C, and a 0.5 M iodine solution in 7.6 ml of THF: pyridine: water (7: 1: 2) was added thereto. The resulting solution was allowed to stand at room temperature for 5 min, and then 3.8 ml of a 2 M Na2S203 aqueous solution was added thereto. After the solution was concentrated until it became a rubber, the The residue was dissolved in 20 ml of dichloromethane and the aqueous layer was extracted with dichloromethane (3x5 ml). The organic layer was collected, washed with a 0.1 M aqueous solution of TEAB (3 x 10 mL), and dried over sodium sulfate. The solution was concentrated and evaporated with toluene (2x10 ml) to remove the remaining pyridine. The residue was dissolved in dichloromethane and purified by silica gel chromatography to provide the title compound (0.5 g, yield: 53%).

NMR 31P (DMSO), 5ppm: -1.03, -0.71

Example _____ 5j _____ Synthesis _____ of _____ 5'-0-dimethoxytrityl-N4-benzoyl-2-0- (t-butyldimethylsilihcitidyl-3'-0-

rcianoetoxifosfinol (3 '> 5'l-2'-0-t-butyldimetillsyliluridine (2e>

30

5'-Dimethoxytrityl-N4-benzoylcytidine-2'-0-t-butyldimethylsilyl-3, - [(2-cyanoethyl) - (N, N-diisopropyl)] phosphoramidite (5b, 1,928 g, 2.00 mmol) and 5'-0-dimethoxytrityl-2'-0-t-butyldimethylsilyluridine (4a, 0.358 g, 1.00 mmol) in 10 ml of anhydrous acetonitrile, and the solution was concentrated until it became gum. 5-Ethylthiotetrazole (0.528 g, 4 mmol, Sigma Aldrich) was dissolved in 20 ml of acetonitrile, and the solution was concentrated until crystals formed. Two solutions were combined using 20 ml of acetonitrile. Then, the combined solution was concentrated to 3 ml and cooled to 0 ° C after 4 hours, and a 0.5 M iodine solution in 12 ml of THF: pyridine: water (7: 1:) was added thereto. 2). The resulting solution was allowed to stand at room temperature for 5 min, and 6 ml of a 2M Na2S203 aqueous solution was added thereto. The solution was concentrated until it became gum. The residue was dissolved in 20 ml of dichloromethane and the aqueous layer was extracted with dichloromethane (3x5 ml). The organic layer was collected, washed with an aqueous solution of 0.1 M TEAB (3 x 10 mL), and dried over sodium sulfate. The solution was concentrated and evaporated with toluene (2 x 10 mL) to remove the remaining pyridine. The residue was dissolved in dichloromethane and purified by silica gel chromatography to provide the title compound (0.868 g, yield: 70%).

NMR 31P (DMSO), 6ppm: -1.05, -0.74 45

Example 6: Synthesis of 5'-0-dimethoxytrityl-N2-isobutyryl-2D-0-ft-butyldimethylsilyltauanosyl-3'-0-

rcianoetoxifosfinol (3 '-> 5'l-2'-0-t-butyldimetillsyliluridine (2fl

S'-dimethoxytrityl-N ^ isobutyrylguanosine ^ '- Ot-butyldimethylsilyl-S' - ^ Z-cyanoethylHN.N-diisopropyl)] 5 phosphoramidite (5c, 3.48 g, 3.59 mmol) and 5'-0 were dissolved -dimethoxytrityl-2'-0-t-but¡ld¡rnet¡ls¡l¡lur¡d¡na (4a, 0.644 g, 1.8 mmol) in 10 ml of anhydrous acetonitrile, and the solution was concentrated until It became rubber. 5-Benzylthiotetrazole (0.379 g, 7.18 mmol) was dissolved in 20 ml of acetonitrile and the solution was concentrated until crystals formed. Two solutions were combined using 20 ml of acetonitrile. Then, the combined solution was concentrated to 3 ml and cooled to 0 ° C after 1.5 hours, and a 0.5 M iodine solution in 22 ml of 10 THF was added thereto: pyridine: water (7 : 1: 2). This solution was allowed to stand at room temperature for 5 min, and an aqueous solution of 2M Na2S203 was added thereto. After the solution was concentrated until it became gum, the residue was dissolved in 20 ml of dichloromethane and the aqueous layer was extracted with dichloromethane (3 x 5 ml). The organic layer was collected, washed with a 0.1 M aqueous solution of TEAB (3 x 10 mL), and dried over sodium sulfate. The solution was concentrated and evaporated with toluene (2x10 ml) to remove the remaining pyridine. The residue was dissolved in dichloromethane and purified by silica gel chromatography to give the title compound (1.126 g, yield: 50%).

NMR 31P (DMSO), 5ppm: -0.52, -0.68

Example 7: Synthesis of 5'-0-dimethoxytrityl-N2-isobutyryl-2D-0- (t-butyldimethylsilyltauanosyl-3'-0-

20 rcianoethoxyphosphinol (3 '-> 5'l-N4-benzoyl-2'-0-t-butyldimethylsilyladenine (2a1

5'-Dimethoxytrityl-N2-isobutyrylguanosine-2'-0-t-butyldimethylsilyl-3 '- [(2-cyanoethyl) - (N, N-diisopropyl)] phosphoramidite (5c, 2.8 g, 2.88 were dissolved mmol) and 5, -0-dimethoxytrityl-N4-benzol-2'-0-t-butyldimethylsilyladenine (4b, 0.7 g, 1.44 mmol) in 10 mL of anhydrous acetonitrile, and the solution was concentrated to That turned into rubber. 5-25 benzylthiotetrazole (1.075 g, 5.6 mmol) was dissolved in 20 ml of acetonitrile and the solution was concentrated until crystals formed. Then, two solutions were combined using 20 ml of acetonitrile. The combined solution was concentrated to 3 ml and cooled to 0 ° C after 1.5 hours, and a solution of 0.5 M iodine in 18 ml of THF: pyridine: water (7: 1: 2) was added thereto. ). This solution was allowed to stand at room temperature for 5 min, and 9 ml of a 2M Na2S203 aqueous solution was added thereto. The solution was concentrated until it became gum. The residue was dissolved in 20 ml of dichloromethane and the aqueous layer was extracted with dichloromethane (3x5 ml). The organic layer was collected, washed with a 0.1 M aqueous solution of TEAB (3 x 10 mL), and dried over sodium sulfate. The solution was concentrated and evaporated with toluene (2x10 ml) to remove the remaining pyridine. The residue was dissolved in dichloromethane and purified by silica gel chromatography to give the title compound (2.182 g, yield: 96%).

35 NMR 31P (DMSO), 6ppm: -0.69, -0.81

Example III Synthesis of phosphoramidite RNA dimer

[Reaction scheme 3]

RIVER — i Bi

River

Bi

L / ° \

/

/

or or2

í

RO --- ~ p = 0

image11

P [N (iPr) 3 | 2 (OCÍi2CH2CN)

ETT

OR OR2 RO — P “0 O—

or,

B,

\

40

NC (H2C) 20

or or2

] p

/ ^ N (¡Pr) 2

Synthesis of ribonucleotide dimers of phosphoramidite RNA UU, CU, GU and GA (1a to 1d).

Ri = DMTr, R2 = TBDMS, R = 2-cyanoethyl, 1a-Bi = U, B2 = U; 1b-Bi = bzC, B2 = U; 1c-Bi, = ibG, B2 = U; 1 d-Bi = ibG; B2, = bzA; 2d-Bi = U; B2 = U, 2e-Bi = bzC, B2 = U, 2f-Bi = ibG, B2 = U, 2g-Bi = ibG, B2 = bzA.

5 Example 8: Synthesis of 5'-0-d¡metox¡tr¡t¡lPc¡anoet¡lfosfor¡l-2'-0- (t-but¡ld¡met¡ls¡l¡hur¡d¡l -3'-0-r (NN- diisopropylaminocyanoethoxyphosphinol (3 '-> 5'1-2'-0-t-butyldimethylsilyluridine (1aí

Compound 2d of Example 4 (0.503 g, 0.44 mmol) and 5-eti III-tetrazole (0.074 g, 0.57 mmol) were dissolved in 10 ml of acetonitrile and the solution was concentrated. 10 ml of acetonitrile was placed in a reaction flask which was then filled with argon, and bis- (diisopropllamine) -2-cyanoethoxy phosphine (0.17 ml, 0.57 mmol) was added dropwise ). The reaction solution was concentrated to approximately 1 ml, allowed to stand for 2 hours and then completely concentrated. The residue was dissolved in 10 ml of dichloromethane and saturated with saturated NaHC03 aqueous solution. The organic layer was washed with saturated NaHC03 aqueous solution (5 x 20 mL) and dried over sodium sulfate. The reaction solution was completely concentrated and water was added until the solution became cloudy. Purification was carried out using an RP18 LiChroprep resin (Merck & Co., Inc., USA) to give the title compound (0.4 g, yield: 70%).

NMR 31P (DMSO), ppm: -138.4, -148.9, -1.03, -0.73

Example 9: Synthesis of 5'-0-dimethoxytrityl-N4-benzoyl-P-cyanoephosphoryl-2'-0- (t-butyldimethylsilihcitidyl-3'-0-r (NN-20 diisopropylaminolcianoethoxyphosphinol (3 '-> 5'í-2'-0-t-butyldimethylsilyluridine Mbl

Compound 2e of Example 5 (0.868 g, 0.70 mmol) and 5-eti III-tetrazole (0.11 g, 0.84 mmol) were dissolved in 10 ml of acetonitrile and the solution was concentrated. 10 ml of acetonltrl were placed in a reaction flask which was then filled with argon, and bis- (diisopropllamlan) -2-canoethoxy phosphine (0.17 ml, 0.57 mmol) was added dropwise. The reaction solution was concentrated to about 1 mL, allowed to stand for 4 hours and then fully concentrated. The residue was dissolved in 10 ml of dichloromethane and saturated with saturated NaHCC aqueous solution> 3. The organic layer was washed with saturated aqueous NaHCOs solution (5 x 20 mL) and dried over sodium sulfate. The reaction solution was completely concentrated and water was added until the solution became cloudy. Purification was carried out using an RP18 LiChroprep resin to give the title compound (0.58 g, yield: 60%).

31P NMR (DMSO), ppm: -148.7, -143.9, -1.18, -0.77

Example 10: Synthesis of 5'-0-d -methoxytrityl-N2-isobutyryl-P-cyanoethylphosphoryl-2'-O- (t-butyldimethylsilihauanosyl-3'- O-IYN-N-diisopropylaminolcianoethoxyphosphinol (3 '-> 5'1 -2'-0-t-butyldimethylsilyluridine (1c)

35

Compound 2f of Example 6 (1,126 g, 0.09 mmol) and 5-etlllotetrazole (0.15 g, 1.17 mmol) were dissolved in 10 ml of acetonitrile and the solution was concentrated. 10 ml of acetonltrl were placed in a reaction flask which was then filled with argon, and bis- (dlopropylamine) -2-cyanoethoxy phosphine (0.35 ml, was added dropwise) 1.17 mmol). The reaction solution was concentrated to about 1 ml, allowed to stand for 4 hours and then completely concentrated. The residue was dissolved in 10 ml of dichloromethane and saturated with aqueous solution of

NaHCC> 3 saturated. The organic layer was washed with saturated NaHC03 aqueous solution (5 x 20 mL) and dried in

sodium sulfate. The reaction solution was completely concentrated and water was added until the solution became cloudy. Purification was carried out using an RP18 LiChroprep resin to give the title compound (0.75 g, yield: 57%).

45 NMR 31P (DMSO), ppm: -150, -148.9, -0.66, -0.49

Example 11: Synthesis of 5'-0-d¡metox¡tr¡t¡l-N2-¡sobut¡r¡lPc¡anoet¡lfosfor¡l-2'-0- (t-but¡ld¡met¡ls ¡Lhahanos¡l-3'- 0-r (NN-diisopropylamino1cyanoethoxyphosphinol (3 '-> 5'1-N4-benzoyl-2'-0-t-butyldimethylsilyladenine (1 cñ

Compound 2g of Example 7 (1.5 g, 1.09 mmol) and 5-ethylthiotetrazole (0.18 g, 1.42 mmol) were dissolved in 10 ml of acetonitrile and the solution was concentrated. 10 ml of acetonitrile was placed in a reaction flask which was then filled with argon, and bis- (diisopropylamino) -2-cyanoethoxy phosphine (0.35 ml, 1.17 mmol) was added dropwise. The reaction solution was concentrated to about 1 mL, allowed to stand for 4 hours and then completely concentrated. The residue was dissolved in 10 ml of dichloromethane and saturated with aqueous solution of

55 NaHC03 saturated. The organic layer was washed with saturated NaHC03 aqueous solution (5 x 20 mL) and dried in

sodium sulfate. The reaction solution was completely concentrated and water was added until the solution became cloudy. Purification was carried out using an RP18 LiChroprep resin to give the title compound (1.081 g, yield: 63%).

NMR 31P (DMSO), ppm: -150, -148.8, -0.63, -0.41

[Reaction scheme 4]

image12

image13

5 Synthesis of UGpA RNA trinucleotides (6a) and CGPA (6b)

R-i = o-chlorophenyl, R = 2-cyanoethyl, R2 = TBDMS. 6a-Bi = U, 6b-Bi = bzC

Example 12: Synthesis of 5'-0-d¡metox¡tr¡t¡l-2'-0- (t-but¡ld¡met¡ls¡l¡hur¡d¡n-3'-¡l- chlorophen¡lfosfat-5'-¡l-N2-¡sobut¡r¡l- 10 2D-0- (t-but¡ld¡met¡ls¡l¡hauanosn-3D-¡l-N4-benxo¡ l-2'-0-t-but¡ld¡met¡ls¡l¡laden¡n-5'-¡lc¡anoet¡lfosfato (6a)

Stage 1: Synthesis of N2-¡sobut¡r¡l-2'-0- (t-but¡ld¡met¡ls¡linauanos¡n-3'-¡l-chlorofen¡l-fosfat-5'-¡ l-N4-benzol-2'-0-t-butyldimethylsilyladenine (2i

15 9 ml of 4% benzenesulfonic acid was added to a solution of dichloromethane: methanol (7: 3) which was cooled to 0 ° C. The solution was added to compound 2c (1,265 g, 0.88 mmol), dissolved in 9 ml of a dichloromethane: methanol solution (7: 3) and allowed to stand at 0 ° C for 3 min. 25 ml of a saturated aqueous NaHC03 solution were added thereto, and the organic layer was washed with a saturated NaHCC aqueous solution> 3, dried over sodium sulfate and concentrated. The residue was purified by silica gel chromatography to give the title compound (0.62 g, yield: 63%).

Stage 2: Synthesis of 5'-0-d¡metox¡tr¡t¡l-2'-0- (t-but¡ld¡met¡ls¡l¡hur¡d¡n-3'-¡l- Chlorophen¡lfosfat-5'-¡l-N2-¡sobut¡ril-2D- 0- (t-but¡ld¡met¡ls¡Mhauanos¡n-3D-¡l-N4-benzoM-2'-0- t-butild¡metMs¡l¡laden¡n-5'-Mc¡anoet¡lfosfato (6a)

Phosphoramldlta U (5a, 0.335 g, 0.39 mmol) and the compound of step 1 (2h, 0.292 g, 0.26 mmol) were dissolved in

anhydrous acetonitñlo and the solution was concentrated. A reaction flask was filled with argon gas and added, to the

same, 10 ml of anhydrous acetonitñlo. 5-Benzltiotetrazole (0.15 g, 0.78 mmol) was dissolved in 10 ml of acetonitol

anhydrous and the solution was concentrated until crystals formed, and then added to the solution

nucleoside The reaction solution was concentrated to approximately 3 ml and allowed to stand for 2 hours. The

The reaction solution was cooled to 0 ° C, and a 0.5 M iodine solution in 2.4 ml of was added thereto.

THF: pyridine: water (7: 1: 2). The resulting solution was allowed to stand at room temperature for 5 min, and was

1.2 ml of an aqueous Na2S2C solution> 3 2 M was added to the solution. After the solution was concentrated

until it became rubber, the residue was dissolved in 20 ml of dichloromethane, and the organic layer was washed with a

0.1 M TEAB aqueous solution. The aqueous layer was extracted with dichloromethane (3 x 5 mL). The organic layer is

35 collected, washed with a 0.1 M aqueous solution of TEAB (3 x 10 mL), and dried over sodium sulfate. The solution is

concentrated and evaporated with toluene (2 x 10 ml) to remove the remaining pyridine. The residue was dissolved in

dichloromethane and purified by silica gel chromatography to provide the title compound (0.413

g yield: 84%).

NMR 25 * * * * 30 31 * * * 35 * * * * 40P (DMSO), OPPM: -6.3, -1.4

40

Example 13: Synthesis of 5'-0-dimethoxytrityl-N4-benzoyl-2'-0- (t-butyldimethylsilihcitidin-3'-yl-chlorophenylphosphat-5'-yl N-isobutyryl-2n-0- (t-butyldimethylsilihauanosin- 3D-il-N4-benzoM2'-0-t-butyldimethylsilyladenin-5'-yl-cyanoethyl phosphate (6b)

5 Phosphoramidite rC (5b, 0.409 g, 0.42 mmol) and the compound from step 1 of Example 12 (2h, 0.238 g, 0.21 mmol) were dissolved and concentrated in anhydrous acetonitrile. A reaction flask was filled with argon gas and 10 ml of anhydrous acetonitrile was added thereto. 5-Benzothiotetrazole (0.123 g, 0.64 mmol) was dissolved in 10 ml of anhydrous acetonitrile and the solution was concentrated until crystals formed, and then added to the nucleoside solution. The reaction solution was concentrated to approximately 3 ml and allowed to stand for 3 hours. The reaction solution was cooled to 0 ° C, and a solution of 0.5 M iodine in 2.6 ml of THF: water: water (7: 1: 2) was added thereto. . The resulting solution was allowed to stand at room temperature for 5 min, and then 1.3 ml of a 2M Na2S203 aqueous solution was added thereto. After the solution was concentrated until it became gum, the The residue was dissolved in 20 ml of dichloromethane, and the organic layer was washed with an aqueous solution of 0.1 M TEAB. The aqueous layer was extracted with dichloromethane (3 × 5 ml). The organic layer was collected, washed with a 0.1 M aqueous solution of TEAB (3 x 10 mL), and dried over sodium sulfate. The solution was concentrated and evaporated with toluene (2x10 ml) to remove the remaining pyridine. The residue was dissolved in dichloromethane and purified by silica gel chromatography to provide the title compound (0.298 g yield: 71%).

NMR 31P (DMSO), ppm: -6.3, -1.2

twenty

Example V: Synthesis of phosphoramidite ribonucleotide trimer

[Reaction scheme 5]

DMTO

image14

RO — P — O

image15

image16

image17

bzA

0 OR2

one

R10 ~ P == 0

or

or.

bzA

OH OR2

OR OR2

NC (H2C) 20 N (iPr} 2

Synthesis of phosphoramidite ribonucleotide trimer (7a and 7b)

Ri = o-chlorophenyl, R = 2-cyanoethyl, R2 = TBDMS. 6a, 7a-B1 = U, 6b, 7b-Bi = bzC

Example 14: Synthesis of 5'-0-dimethoxytrityl-2'-0- / t-butyldimethylsilihuridin-3'-yl-chlorophenylphosphat-5'-yl-N2-isobutyryl-2D-0- (t-butyldimethylsilihauanosin-3D-il -N4-benzoyl-2'-0-t-butyldimethylsilyladenin-5'-yl-cyanoethylphosphate-3-0- (2 ^ cyanoethyl-NN-diisopropylphosphoramidite (7a)

5 The compound 6a of Example 12 (0.41 g, 0.21 mmol) and 5-ethylthiotetrazole (0.037 g, 0.57 mmol) were dissolved and concentrated in 10 ml of acetonitrile. 10 ml of anhydrous acetonitrile was placed in a reaction flask which was then filled with argon, and bis- (diisopropylamino) -2-cyanoethoxy phosphine (0.084 ml, 0.28 mmol) was added dropwise. The reaction solution was concentrated to approximately 1 ml, allowed to stand for 4 hours, followed by the complete concentration. The residue was dissolved in 10 ml of dichloromethane and saturated with saturated aqueous NaHC03 solution. The organic layer was washed with saturated aqueous NaHCOs solution (5 x 20 mL) and dried over sodium sulfate. The reaction solution was completely concentrated and water was added until the solution became cloudy. Purification was carried out using an RP18 LiChroprep resin to give the title compound (0.319 g, yield: 72%).

NMR 31P (DMSO), 6ppm: -150.2, -148.9, -6, -1.4 15

Example 15: Synthesis of 5'-0-dimethoxytrityl-N4-benzoyl-2'-0- (t-butyldimethylsilylcithidin-3'-yl-chlorophenylphosphat-5'-yl N-isobutyryl-2D-0- (t-butyldimethylsilyltatanososin- 3D-il-N4-benzoyl-2'-0-t-butyldimethylsilyladenin-5'-yl-cyanoethylphosphate-3-Q- (2-cyanoethyl-NN-diisopropylphosphoramidite (7bl

The compound 6b of Example 13 (0.298 g, 0.15 mmol) and 5-ethylthiotetrazole (0.025 g, 0.20 mmol) were dissolved and concentrated in 10 ml of acetonitrile. 10 ml of anhydrous acetonitrile was placed in a reaction flask which was then filled with argon, and bis- (diisopropylamino) -2-cyanoethoxy phosphine (0.06 ml, 0.20 mmol) was added dropwise thereto. The reaction solution was concentrated to approximately 1 ml and allowed to stand for 4 hours, followed by the complete concentration. The residue was dissolved in 10 ml of dichloromethane and saturated with saturated aqueous NaHCOs solution. The organic layer was washed with saturated NaHC03 aqueous solution (5 x 20 mL) and dried over sodium sulfate. The reaction solution was completely concentrated and water was added until the solution became cloudy. Purification was carried out using an RP18 LiChroprep resin to give the title compound (0.188 g, yield: 60%).

NMR 31P (DMSO), ppm: -150.2, -148.9, -6, -1.0 30

Example VI: Synthesis of siRNA using phosphoramidite RNA dimer

All siRNAs were synthesized using a Polygen DNA / RNA synthesizer at a scale of 0.8 pinol in its trityl-off form. The 3'-terminal end used CPG RNA. CPG RNA (Glen Research) was used in an amount of 30 pmmol / g loading, and the monomer bases were respectively rA * 33, rC * 33, rGtac and U (Proligo) phosphoramidites. The phosphoramidite monomer and dimer were used in the form of a 0.05 M solution in acetonitrile. The equivalents of the monomers and dimers were respectively 2.5 equivalents per cycle. One activator was 0.5 M 5-ethylthi-tetrazole (in acetonitrile). Solid supports and protecting groups were deprotected by heating the reaction solution at 65 ° C for 2 hours using a mixture of aqueous ammonia and ethanol (3: 1), and solution 40 was lyophilized. The residue was dissolved in 0.4 ml of a solution of N-methylpyridone: triethylamine: triethylamine trihydrofluoride (6: 3: 4) and heated at 65 ° C for 2 hours. 4 ml of n-butyl alcohol was added to the resulting solution, which was then cooled in a refrigerator for 2 hours and centrifuged to obtain solid siRNA, followed by lyophilization. The yield of the raw siRNAs was quantitatively analyzed using a UV spectrophotometer at 260 nm and the purity thereof was analyzed by reverse phase HPLC. The coefficients of extinction of naturally occurring ribonucleotides for the calculation of the concentration are the following: rA, 15400: rC, 7200: U, 9900: and rG, 11500. A molecular weight of each siRNA was confirmed by mass analysis using MALDI-TOF (Bruker, Autoflex).

Example 16: Synthesis of siRNA GFP sense using the GU RNA dimer

fifty

The siRNA sense GFP had a sequence of 5'-GUU CAG CGU GUC CGG CGA GLJU-3D (SEQ ID No. 1). The synthesis of siRNA was carried out analogously to Example 15, and the coupling period for the GU dimer and the monomers was 10 min each. The dimer used for the first coupling stage was GU, to which monomer units were then attached. The purity of the product was measured by reverse phase chromatography, and the analyzer used was an Agilent 1100 system. The chromatography buffer was a mixture of 100 nM of TEAA (pH 7.0) and acetonitrile. The purity and yield of the siRNA product were compared with those of the GFP sense siRNA that was obtained using the monomer instead of the dimer as the first ribonucleotide. The results are given below in Table 1.

60 [Table 1]

 First ribonucleotide

 Purity of siRNA

 Monomer

 52%

 Dimer

 73%

The GFP antisense siRNA had a sequence of 5'-CUC GCC GGA CAC GCU GAA CUU-3 '(SEQ ID No. 2). The synthesis of siRNA was carried out analogously to Example 15, and the coupling period for the CU 5 dimer and the monomers was 10 min each. The dimer used for the first coupling stage was CU, to which monomer units were then attached. The purity of the product was measured by reverse phase chromatography, and the analyzer used was an Agllent 1100 system. The chromatography buffer was a mixture of 100 nM of TEAA (pH 7.0) and acetonitrile. The purity and yield of the siRNA product were compared with those of the GFP antlsentldo siRNA that was obtained using the monomer instead of the dimer as the first ribonucleotide. The 10 results are given below in Table 2.

[Table 2]

 First ribonucleotide

 Purity of siRNA

 Monomer

 63%

 Dimer

 78%

Example 18: Synthesis of JNK antisense siRNA using the RNA dimer

The JNK antisense siRNA had a sequence of 5'-AGA AGG UAG GAC AUU CUU UUU-3 '(SEQ ID No. 3). The synthesis of siRNA was carried out analogously to Example 15, and the coupling period for the UU dimer and monomers was 10 min each. The dimer used for the first coupling stage was UU, to which 20 monomer units were then attached. The purity of the product was measured by reverse phase chromatography, and the analyzer used was an Agilent 1100 system. The chromatography buffer was a mixture of 100 nM of TEAA (pH 7.0) and acetonitrile. The purity and yield of the siRNA product were compared with those of the JNK antisense siRNA that was obtained using the monomer instead of the dimer as the first ribonucleotide. The results are given below in Table 3.

25

[Table 3]

 First ribonucleotide

 Purity of siRNA

 Monomer

 71%

 Dimer

 88%

Example 19: Synthesis of siRNA siRNA using the GA RNA dimer

30

The siRNA sense SEI had a sequence of 5-GCAAGG GUC UGAAGC GGAA-3 (SEQ ID N ° 4). The synthesis of siRNA was carried out analogously to Example 15, and the coupling period was 10 min and 15 min for the monomers and the GA dimer, respectively. The dimer used for the first coupling stage was GA, to which monomer units were then attached. The purity of the product was measured by reverse phase chromatography, and the analyzer used was an Agllent 1100 system. The chromatography buffer was a mixture of 100 nM of TEAA (pH 7.0) and acetonitrile. The purity and yield of the siRNA product were compared with those of the SEI sense siRNA that was obtained using the monomer instead of the dimer as the first ribonucleotide. The results are given below in Table 4.

40 [Table 4]

 First ribonucleotide

 Purity of siRNA

 Monomer

 65%

 Dimer

 78%

Although preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope of the invention as disclosed in the appended claims.

Industrial applicability

The present invention allows efficient synthesis of high speed and high purity of ribonucleotide ollomers. The method of the present invention provides a ribonucleotide ollomer having a purity of 15-20% greater than that of the conventional technique.

Claims (8)

1. A method of preparing ribonucleotide oligomers, comprising: 5 (a) coupling a ribonucleotide dimer or ribonucleotide trimer to a ribonucleoside attached to solid supports or to universal solid supports as starting material; (b) sequentially coupling ribonucleotide monomers to the structures resulting from step (a) to prepare ribonucleotide oligomers; Y (c) remove ribonucleotide oligomers from solid supports. 10 wherein the ribonucleotide dimer in step (a) is represented by Formula 5: image 1 or or2 R3O — p = o image2 (5) fifteen in which each R1, R2, R3 and Rs are independently protecting groups, each B1 and B2 are independently 0 (CH2) 2CN -p \. • N (iPr) 2 nucleoside bases, and R6 is hydrogen or in which iPr is isopropyl; wherein the ribonucleotide trimer in step (a) is represented by Formula 6: R-jO- B3 \ _ or or2 RoO — P = 0 \ or. B2 Or 0R4 RcO — P = 0 image3 OR7OR6 (6) in which each R1, R2, R3, R4, R5 and R6 are independently protecting groups, each B1, B2 and B3 are independently nucleoside bases, and R7 is hydrogen or 0 (CH2) 2CN Y XN (iPr) 2 in which Pr is isopropyl. The method of claim 1, wherein the method includes: (a) coupling a ribonucleotide dimer according to claim 1 to a ribonucleoside attached to solid supports or to universal solid supports as starting material; (b) sequentially coupling ribonucleotide monomers to the structures resulting from step (a) to prepare ribonucleotide oligomers; Y (c) remove ribonucleotide oligomers from solid supports. 3. The method of claim 1, wherein the method includes: (A) coupling a ribonucleotide trimer according to claim 1 to a ribonucleoside attached to solid supports or to universal solid supports as starting material; (b) sequentially coupling ribonucleotide monomers to the structures resulting from step (a) to prepare ribonucleotide oligomers; Y (c) remove ribonucleotide oligomers from solid supports. twenty 4. The method of any one of claims 1 to 3, wherein the ribonucleotide monomer is a ribonucleoside phosphoramidite. 5. The method of any one of claims 1 to 3, wherein the carbononucleotide ollomer is one that contains at least one ribonucleotide selected from 2-O-halogen ribonucleotide, 2-amino ribonucleotide, 2'-0-alkyl ribonucleotide and 2'-0-alkoxy ribonucleotide. The method of any one of claims 1 to 3, wherein the ribonucleotide oligomer has a phosphodiester, phosphoramidate, alkylphosphoramidate, alkyl phosphonate, phosphorothioate, alkylphosphotriester or alkylphosphonothioate linkage. 7. The method of claim 6, wherein the ribonucleotide oligomer has a phosphodiester or phosphoramidate bond. 0 8. The method of claim 1 or 2, further comprising the step of preparing a ribonucleotide dimer of step (a) by a reaction that includes the coupling of a compound of Formula 1 with a compound of Formula 2: image4 or or2 r3o — p = o OR- (one) In which each Ri, R2, and R3 are independently protecting groups, and B2 is a nucleosphidic base; R40 ----- / ° \ | OH OR5 (2) wherein each R4 and R5 are independently protective groups, and B1 is a nucleosphidic base. 9. The method of claim 1 or 2, further comprising preparing the ribonucleotide dimer of step (a) 20 by a reaction that includes the coupling of a compound of Formula 3 with a compound of Formula 2: R-jO— OR Be OR OR2 TO R31 ^ 32 (3) wherein each R1 and R2 are independently protecting groups, R31 is 2-cyanoethoxy, cyanomethoxy, 4-cyano-2- butenyloxy or diphenylmethylsilylethoxy, R32 is diakylamino, and B2 is a nucleosphidic base; R40— ^ image5 OH OR5 (2) in which each R4 and R5 are independently protective groups, and B1 is a nucleobase. 10. The method of claim 1 or 3, further comprising preparing the ribonucleotide trimer of step (a) 5 by a reaction that includes: (a) reacting a ribonucleotide dimer of Formula 4 with an acid to remove R1 of Formula 4; Y (b) coupling the product resulting from step (a) with a 3'-phosphoramidite ribonucleoside to prepare a ribonucleotide trimer: wherein each R-i, R2, R3 and R5 are independently protecting groups, and each B1 and B2 are independently nucleoside bases. The method of claim 10, wherein the acid of step (a) is benzenesulfonic acid. image6 image7 or or2 OR ------- Bi or T image8 OH ORs (4) REFERENCES CITED IN THE DESCRIPTION This list of references cited by the applicant is solely for the convenience of the reader. It is not part of the European patent document. Despite the care taken in the collection of references, errors or omissions cannot be excluded and the EPO denies any responsibility in this regard. Patent documents cited in the description 10 * US 5149798 A [0003] • WO 0220543 A [0017] Different patent literature cited in the description fifteen • KHORANA et al. J. Molec. BioL, 1972. vol. 72, 209 [0003] • REESE, Tetrahedron Lett., 1978, vol. 34, 3143 twenty • BEAUCAGE; CARUTHERS Tetrahedron Lett., 1981, vol. 22, 1859 [0003] • AGRAWAL; GOODCHILD Tetrahedron Lett., 1987, vol. 28, 3539 [0003] 25 • CONNOLLY et al. Biochemistry, 1984, vol. 23, 3443 [0003] • JAGER et al. Biochemistry 1988, vol. 27, 7237 [0003] 30 • AGRAWAL et al. Proc. Nati Acad. Sci. USA, 1988, vol. 85, 7079 [0003] • METHODS IN MOLECULAR BIOLOGY. Protocols for Oligonucleotides and Analogs. Humana Press, 35 1993, vol. 20, 63-80 [0003] • Methods in Molecular Biology. Prolocolsfor Oligonucleotide Conjugates. Humana Press, 1994, vol. 26 [0003] • Oligonucleotides and Analagues: A Practical Ap- 40 proach IRL Press, 1991,155-183 [0003] • Antisense Res. And Applns. CRC Press 1993, 375 [0003] • Gene Regulation: Biology of Anlisense RNA and 4c DNA. Raven Press, 1992 [0003] • US 4458066 A [0038] • US 4415732 A [0038] BARRER et al. Proc. Nati Acad. Sci. USA, 1996, vol. 93, 514 [0004] AGRAWAL et al. Proc. Nati Acad Sci. USA, 1988, vol. 85, 7079 [0004] LETTER et al. Proc. Nati Acad Sci. USA, 1990, vol. 87, 3420-3434 [0004] OFFENSPERGER et al. EMBO J., 1993, vol. 12, 1257 [0004] SHEN Cet al. FEBS Lett., 2003, vol. 539 (1-3), 111-4 [0005] FIRE A et al. Nature, 1998. vol. 391 (6669), 806-11 [0005] ELBASHIR, S.M. state. Nature, 2001, vol. 411 (6836), 494-8 [0005] MOULDY SIOUD. Therapeutic siRNAs. Trends in pharmacoiogicai Sciences, 2004, 22-28 [0006] KROTZ A.H. et al. Bioorganic Medicinal Chemistry Letters, 1997, vol. 7 (1), 73-78 [0013] ELEUTERI Aetal. Nucieosides & Nucleotides, 1999, vol. 18 (3), 475-483 [0014] CARUTHERS et al. Genetic Engineering, 1982, vol. 4, 1-17 [0038] Users Manual Model 392 and 394 Polynucleotide Synthesizers. Applied Biosystems, 1991, 6-1, 6-22 [0038]